Measuring the HIV reservoir

For researchers working to develop an HIV cure, it is important to have some way of measuring the effects of a potential therapy. In particular, there is a need to accurately quantify the pockets of virus (latent HIV reservoir) that persist in the body after ART suppresses viral load to undetectable levels. There are a variety of technologies available that are currently employed in studies, but each has pros and cons.

The “gold standard” test for assessing the size of the latent HIV reservoir is called the quantitative virus outgrowth assay (qVOA). Performing qVOA requires taking a large blood sample (in the range of 120–180 ml) and extracting resting CD4 T cells, which are then activated in the laboratory in order to induce HIV production by any latently infected cells that are present. The amount of HIV generated is measured, and a statistical approach used to calculate the number of latently infected CD4 T cells that were in the sample—the result is expressed as infectious units per million cells (IUPM). The test takes a total of 14 days to perform.

The advantages are that qVOA measures latent HIV that is capable of replicating (described as “replication-competent”)—this is important because it has been shown that the majority of HIV DNA that can be found integrated into the DNA of resting CD4 T cells is mutated in ways that render it defective. These defective HIV DNA copies cannot produce new viruses capable of infecting other cells.

The disadvantages of qVOA include the requirement for large volumes of blood and the length of time it takes to perform, as well as the cost (approximately $1,000). Additionally, it has recently been discovered that activating resting CD4 T cells—the key step in the test—does not induce HIV production by all the replication-competent latent HIV that is present in the sample. It is now estimated that there is likely about 60–70 times more replication-competent latent HIV than is detected by qVOA.

At least two improved variants of the qVOA that are cheaper and require less blood volume have more recently been described—including a new assay that may address the problem of underestimating the frequency of latently infected cells (developed by Anwesha Sanya and colleagues at the University of Pittsburgh)—but these new approaches have yet to undergo extensive evaluation.

The Tat/Rev-induced limiting dilution assay (TILDA) is a relatively new (but increasingly popular) approach to quantifying the HIV reservoir. TILDA has some similarities to qVOA but measures particular forms of HIV RNA after CD4 T cells are activated in the laboratory—it is thought to preferentially capture replication-competent HIV but may also pick up some defective viruses.

Several more straightforward tests look for HIV genetic material in different ways. The most common measures levels of HIV DNA in a sample using polymerase chain reaction (PCR), but this technique cannot distinguish between defective and replication-competent HIV. A more complicated adaptation of this approach can specifically quantify HIV DNA that is integrated into the genome of CD4 T cells, but also cannot ascertain if it is replication-competent or not. Nevertheless these tests are frequently employed to give a rough idea of the size of the HIV reservoir.

Attempts to accurately measure the HIV reservoir are complicated by the fact that the vast majority of latently infected cells are known to reside in lymph tissues rather than the blood. For this reason some studies look to collect lymph biopsies, and new technologies are being developed to better quantify the amount of virus in tissues. Sampling from the central nervous system (CNS) is another challenge, considered important due to evidence that HIV may persist in the brain. There is uncertainty regarding whether HIV can become latent in cell types other than CD4 T cells, such as macrophages, and whether additional tests may be needed to evaluate the contribution of non-CD4 cells to the reservoir—ongoing research is aiming to answer this question.

An overarching problem with HIV reservoir measurement is highlighted by the cases of temporary remission described in “The Challenge of Defining an HIV Cure”. In these individuals, the HIV reservoir could not be detected by any current test, even for a period after an ART interruption. But we know from the eventual viral load rebound that latently infected cells were still present somewhere in their bodies. This limitation in the ability of scientists to detect small HIV reservoirs is a big part of the reason why ART interruptions are still considered important in some cure research studies (see “Time Out”).